Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map

Inter-simple sequence repeat (ISSR) analysis was evaluated for its usefulness in generating markers to extend the genetic linkage map of Citrus using a backcross population previously mapped with restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and isozyme markers. ISSR markers were obtained through the simple technique of PCR followed by analysis on agarose gels, using simple sequence repeat (SSR) primers. Optimization of reaction conditions was achieved for 50% of the SSR primers screened, and the primers amplified reproducible polymorphic bands in the parents and progeny of the backcross population. Mendelian segregation of the polymorphic bands was demonstrated, with an insignificant number of skewed loci. Most of the SSR primers produced dominant loci; however co-dominance was observed with loci derived from three primers. A new genetic map was produced by combining the segregation data for the ISSR markers and data for the RFLP, RAPD and isozyme markers from the previous map and creating genetic linkages among all the markers using JoinMap 2.0 mapping software. The new map has an improved distribution of markers along the linkage groups with fewer gaps, and marker order showed partial or complete conservation in the linkage groups. The incorporation of ISSR markers into the genetic linkage map demonstrates that ISSR markers are suitable for genetic mapping in Citrus. Received: 3 February 2000 / Accepted: 12 May 2000     

Isolation and characterization of 16 microsatellite loci in an endangered fish Ussuri cisco, <Emphasis Type="Italic">Coregonus usssruensis</Emphasis>

Sixteen polymorphic microsatellite loci were isolated from an enriched genomic library of Ussuri cisco, Coregonus usssruensi. The polymorphism of these loci was tested on a population of 30 individuals captured from Heilongjiang River. The number of observed alleles per locus ranged from 3 to 18. The expected and observed heterozygosity values ranged from 0.126 to 0.919 and 0.133 to 0.833, respectively. Five loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. No significant linkage disequilibrium was detected. These polymorphic markers will be used to assess population structure in Ussuri cisco.     

Genetic variation,population structure and identification of yellow catfish, <Emphasis Type="Italic">Mystus nemurus</Emphasis> (C&;V) in Thailand using RAPD,ISSR and SCAR marker

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.     

Development of microsatellite marker loci for European hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis> L.) from ISSR fragments

Polymorphic microsatellite markers were developed for European hazelnut (Corylus avellana L.) from the sequences of inter-simple sequence repeat (ISSR) fragments and flanking regions. Twenty-five ISSR primers were used to generate fragments for cloning. Of the 520 unique sequences obtained, 41 contained long internal repeats (≥20 bp) with flanking sequences sufficient for primer design. From these, we developed 23 new polymorphic microsatellite loci. The flanking sequences were obtained for fragment ends by chromosome walking, and an additional 47 polymorphic markers were developed. Two additional polymorphic markers were developed from a GA-enriched library. The 72 new marker loci were characterized using 50 diverse hazelnut accessions. For the internal repeat loci, the number of alleles per locus ranged from 2 to 16, with a mean of 7.52. Mean values for expected heterozygosity (H_e), observed heterozygosity (H_o), and polymorphism information content (PIC) were 0.62, 0.59, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.05 at six of the 23 loci. For the 47 marker loci developed from fragment ends, the number of alleles per locus ranged from 2 to 16, with a mean of 7.30. Mean values for H_e, H_o, and PIC were 0.62, 0.47, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.10 at 18 of the 47 loci. Of the 70 loci developed from ISSR and flanking sequences, 50 segregated in our mapping population and were assigned to linkage groups.     

65份野生油茶种质遗传多样性的ISSR和RAPD标记分析

利用ISSR和RAPD标记,对名邛台地野生油茶种质进行遗传多样性分析。从60条简单重复序列引物中筛选出16条引物,在65份样品中共扩增出213条带,其中多态位点为203个,多态位点百分率为95.31%;从30条寡居核苷酸引物中筛选出8条引物,共扩增出105条带,其中多态性位点94个,多态位点百分率为89.52%。结果表明:名邛台地野生油茶种质具有较丰富的遗传多样性,ISSR和RAPD标记可以应用于油茶种质遗传多样性分析。     

Isolation and characterization of microsatellite loci from the Japanese conger eel Conger myriaster

Five polymorphic microsatellite markers were developed in the Japanese conger eel (Conger myriaster) to understand the recruitment mechanisms and population structure of this species, the spawning sites and migration routes of which are unknown. Three of the loci were highly polymorphic and will serve as powerful tools for detailed population studies of the Japanese conger eel. One of the other two loci showed significant departure from Hardy–Weinberg equilibrium and the other showed low allelic diversity.     

Isolation and characterization of polymorphic microsatellite DNA markers in the rock bream (Oplegnathus fasciatus)

From a (GT)₁₃-enriched genomic library of Oplegnathus fasciatus, 14 polymorphic microsatellite were isolated and characterized in a test population with alleles ranging from two to nine, the observed and expected heterozygosities from 0.0000 to 1.0000, and from 0.1726 to 0.8507, respectively. Five loci deviated from the Hardy–Weinberg equilibrium, and linkage disequilibrium between two loci was significant. Two loci were also polymorphic in Pagrosomus major assessed for cross-species amplification. These polymorphic microsatellites will be useful for genetic diversity analysis and molecule-assisted breeding for O. fasciatus. Changwei Shao contributed equally.     

Comparative analysis of the informativeness of ISSR markers for estimating genetic diversity of horse breeds

The informativeness of six different types of ISSR markers was compared in order to evaluate genetic diversity of Mongolian, Buryat and Tuvinian horse breeds (Equus caballus). The dinucleotide-based ISSR primers ((GA)₉C, (AG)₉C, and (CA)₉G) proved to be of little use to examine genetic polymorphism among horses due to the low number of amplified and polymorphic fragments. The polymorphism information content (PIC) for the dinucleotide-based ISSR primers was 2.88 times lower than that for trinucleotide-based ISSR markers. Among the trinucleotide-based ISSR markers, CAC ISSR marker was also found to be noninformative, due to the low number of polymorphic loci identified. The highest values of the polymorphism information content (PIC) were obtained for two types of ISSR markers, based on (GAG)₆C (PIC, 3.61) and on (ACC)₆G (PIC, 2.50). Thus, the results of the present study showed that GAG and ACC ISSR markers were most informative for evaluation of genetic diversity among horses.     

ISSR as New Markers for Identification of Homokaryotic Protoclones of Agaricus bisporus

To accelerate the breeding of Agaricus bisporus, quick and reliable methods to identify the infrequent homokaryons are necessary. A new marker, inter simple sequence repeat (ISSR) fingerprinting, is described for differentiation of homo- and hetero-karyotic protoclones. Nine slow growing protoclones, two strandy and seven appressed, were analyzed for the first time with ISSR amplifications. The patterns were highly polymorphic and very reproducible. Among 40 primers tested, 7 ISSR primers were selected for the analysis of genomic DNA and generated a total of 68 ISSR fragments. ISSR fingerprinting detected 44.12% polymorphic loci. All appressed homokaryons carried a subset of ISSR markers found in the heterokaryons, and clustered separately in dendrogram. These were not able to produce a fruiting body. A test of cross-fertility and the following fruiting trial proved that 7 of the 9 protoclones with different ISSR fingerprints were homokaryons. These results demonstrated that ISSR markers provide an efficient alternate for identification of homokaryons and suggest these markers be considered as new tools for the survey of Agaricus species.     

Twelve novel polymorphic microsatellite loci for the Yellow grouper (Epinephelus awoara) and cross-species amplifications

Yellow grouper (Epinephelus awoara) is a commercially important marine fish species. A dinucleotide-enriched genomic library of E. awoara was constructed using the method of FIASCO. Twelve loci were polymorphic in a test population with alleles per locus ranging from two to eight, and observed and expected heterozygosities per locus from 0.13 to 1.00 and from 0.20 to 0.86, respectively. Five loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic diversity of E. awoara and related species. L. Zhao and C. Shao have contributed equally to this work.     

Comparison of RAPD and ISSR markers for genetic diversity analysis among different endangered Mangifera indica genotypes of Indian Gir forest region

The genetic variability and relationships among 20 Mangifera indica genotypes representing 15 endangered and 5 cultivars, obtained from Indian Gir forest region, were analyzed using 10 random amplified polymorphic DNA (RAPD) and 21 inter simple sequence repeat (ISSR) markers. RAPD markers were more efficient than the ISSR assay with regards to polymorphism detection. Also, the average numbers of polymorphic loci per primer, average polymorphic information content (PIC) and primer index (PI) values were more for RAPD than for ISSR. But, total number of genotype specific marker loci, Nei’s genetic diversity (h), Shannon’s information index (I), total heterozygosity (Ht), average heterozygosity (Hs) and mean coefficient of gene differentiation (Gst) were more for ISSR as compared to RAPD markers. The regression test between the two Nei’s genetic diversity indexes showed low regression between RAPD and ISSR based similarities but maximum for RAPD and RAPD + ISSR based similarities. The pattern of clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared. Thus, both the markers were equally important for genetic diversity analysis in M. indica.     

利用RAPD和ISSR分子标记分析怀地黄种质遗传多样性

用RAPD与ISSR技术对怀地黄的8个品种和2个脱毒品系进行了种质遗传多样性分析。分别从80条RAPD引物和44条ISSR引物中筛选出适合怀地黄种质分析的17条RAPD引物和10条ISSR引物,用于RAPD和ISSR分析。17条RAPD引物共扩增出177条带, 多态性位点数为109; 多态性位点比率为61.58%;平均多样性指数(I)为0.3135;每个位点的有效等位基因数（Ne）是1.3641; 10条ISSR引物共扩增出110条带. 多态性位点数为79; 多态性位点比率为71.58%;平均多样性指数(I)为0.3577;每个位点的有效等位基因数（Ne）是1.4037。 基于扩增条带数据库建立了各自的Jaccard遗传相关系数矩阵,构建了相似的分子树状图,将10个供试材料分为2类:一类群含组培85.5、大田85.5、组培9302、大田9302、金状元和金白6个材料;另一类群含北京1号、大红袍、地黄9104和野生地黄4个材料。两种分子标记的分析结果呈极显著正相关(r＝0.649)。结果表明,RAPD与ISSR标记适合于怀地黄种质遗传多样性分析,ISSR标记技术是一种多态性和重复性优于RAPD技术的实用技术。     

Molecular genetic diversity of Satureja bachtiarica

Fifty-seven genotypes from eight population of Satureja bachtiarica was evaluated using fifteen ISSR and eleven RAPD markers. DNA profiling using RAPD primers amplified 84 loci, among which 81 were polymorphic with an average of 7.36 polymorphic fragments per locus. Also, using RAPD markers maximum and minimum polymorphic bands observed for Semyrom (77.38 %) and Farsan (40.48 %) populations, respectively. Semyrom population recorded the highest unbiased expected heterozygosity (0.259) and Shannon’s Indices (0.38). While, the lowest values of unbiased expected heterozygosity (0.172) and Shannon’s Index (0.245) were recorded for Eghlid and Farsan populations, respectively. On the other hand, ISSR primers produced 136 bands, from which 134 were polymorphic with an average of 9.06 polymorphic fragments per primer (98.52 %). The ISSR markers evaluation revealed that maximum and minimum polymorphic bands observed for Semyrom (66.18 %) and Farsan (31.62 %), respectively. Shahrekorud population recorded the highest unbiased expected heterozygosity (0.211) and Shannon’s Indices (0.301). While, the lowest value of unbiased expected heterozygosity (0.175) observed for Farsan and Yazd populations and the lowest Shannon’s Index (0.191) recorded by Farsan population. The overall results of the study revealed that both ISSR and RAPD markers were effective for evaluation of genetic variation of S. bachtiarica.     

Polymorphic dinucleotide microsatellites in tongue sole (Cynoglossus semilaevis)

The tongue sole, Cynoglossus semilaevis, is a rare marine flatfish distributed in Chinese coastal waters. From a (GT)_n‐enriched genomic library, 57 microsatellites were isolated and characterized. Seventeen of these loci were polymorphic in a test population with alleles ranging from three to 13, and observed and expected heterozygosities from 0.1613 to 1.0000 and from 0.2126 to 0.8983, respectively. Five loci deviated from the Hardy–Weinberg equilibrium in the sampled population, and linkage disequilibrium between two loci was significant after applying Bonferroni correction. Three additional fish species assessed for cross‐species amplification revealed that only one locus was also polymorphic in one species. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the breeding strategy and investigate the fine‐scale population structure in C. semilaevis.     

Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers

An efficient micropropagation protocol produced large number of plants of the three elite banana (Musa spp.) cultivars Robusta (AAA), Giant Governor (AAA) and Martaman (AAB) from shoot tip meristem. The genetic relationships and fidelity among the cultivars and micropropagated plants as assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers, revealed three somaclonal variants from Robusta and three from Giant Governor. A total of 5330 RAPD and 2741 ISSR fragments were generated with 21 RAPD and 12 ISSR primers in micropropagated plants. The percentage of polymorphic loci by RAPD and ISSR were found to be 1.75, 5.08 in Robusta and 0.83, 5.0 in Giant Governor respectively. Among the two marker systems used, ISSR fingerprinting detected more polymorphism than RAPD in Robusta and Giant Governor with most of the primers showing similar fingerprinting profile, whereas Martaman revealed complete genetic stability.     

Molecular diversity and relationships among <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> cultivars based on inter-simple sequence repeat (ISSR) markers

Spring orchid (Cymbidium goeringii) is a popular flowering plant species. There have been few molecular studies of the genetic diversity and conservation genetics on this species. An assessment of the level of genetic diversity in cultivated spring orchid would facilitate development of the future germplasm conservation for cultivar improvement. In the present study, DNA markers of intersimple sequence repeats (ISSR) were identified and the ISSR fingerprinting technique was used to evaluate genetic diversity in C. goeringii cultivars. Twenty-five ISSR primers were selected to produce a total of 224 ISSR loci for evaluation of the genetic diversity. A wide genetic variation was found in the 50 tested cultivars with Nei’s gene diversity (H = 0.2241) and 93.75% of polymorphic loci. Fifty cultivars were unequivocally distinguished based on ISSR fingerprinting. Cultivar-specific ISSR markers were identified in seven of 50 tested cultivars. Unweighted pair-group mean analysis (UPGMA) and principal coordinates analysis (PCA) grouped them into two clusters: one composed the cultivars mainly from Japan, and the other contained three major subclusters mainly from China. Two Chinese subclusters were generally consistent with horticultural classification, and the third Chinese subcluster contained cultivars from various horticultural groups. Our results suggest that the ISSR technique provides a powerful tool for cultivar identification and establishment of genetic relationships of cultivars in C. goeringii.     

An intersubspecific genetic map of<Emphasis Type="Italic"> Lens</Emphasis>

A Lens map was developed based on the segregational analysis of five kinds of molecular and morphological genetic markers in 113 F₂ plants obtained from a single hybrid of Lens culinaris ssp. culinaris × L. c. ssp. orientalis. A total of 200 markers were used on the F₂ population, including 71 RAPDs, 39 ISSRs, 83 AFLPs, two SSRs and five morphological loci. The AFLP technique generated more polymorphic markers than any of the others, although AFLP markers also showed the highest proportion (29.1%) of distorted segregation. At a LOD score of 3.0, 161 markers were grouped into ten linkage groups covering 2,172.4 cM, with an average distance between markers of 15.87 cM. There were six large groups with 12 or more markers each, and four small groups with two or three markers each. Thirty-nine markers were unlinked. A tendency for markers to cluster in the central regions of large linkage groups was observed. Likewise, clusters of AFLP, ISSR or RAPD markers were also observed in some linkage groups, although RAPD markers were more evenly spaced along the linkage groups. In addition, two SSR, three RAPD and one ISSR markers segregated as codominant. ISSR markers are valuable tools for Lens genetic mapping and they have a high potential in the generation of saturated Lens maps.Communicated by H.C. Becker     

Polymorphism analysis in advanced mutant population of oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.) using ISSR markers

Present investigation was carried out to evaluate genetic diversity among 38 M6 population of oat cv. JO-1. To validate the observed morpho-physiological variations, these lines were analyzed with 21 ISSR primers. A total of 132 loci were amplified by these 21 ISSR markers and 116 loci were found to be polymorphic (87.87 %). The genetic similarity coefficient values among 39 oat genotypes based on ISSR analysis ranged from 0.305 to 0.957. The cluster analysis divided the oat genotypes into two groups. Mutants JMO 81 and JMO 82 were found to be most divergent, hence can be used as parents in breeding program for the development of superior cultivars.     

辣椒种质遗传多样性的RAPD和ISSR及其表型数据分析

用RAPDI、SSR分子标记及28个表型性状数据对辣椒属5个栽培种的13份材料进行了分析,结果表明:23条RAPD引物共扩增出209条带,平均每个引物扩增出9.09条,多态性位点比率为83.73%;16条ISSR引物共扩增出94条带,平均每个引物扩增出5.88条,多态性位点比率为79.79%.与RAPD相比,ISSR标记检测到的有效等位基因数(Ne)及Shannon多样性指数(I)、遗传离散度(Ht)和遗传分化系数(Gst)等遗传多样性参数都较大,多态性位点比例在亲缘关系较近的一年生辣椒(Capsicum annuum)种内较高,说明ISSR有更高的多态性检测效率,并且适合亲缘关系较近的种群间遗传多样性分析.基于RAPDI、SSR的聚类与基于表型数据的聚类之间存在极显著正相关,且都能将C.annuum与其它栽培种区分开来.     

枫香自然种群遗传多样性的ISSR分析

利用ISSR分子标记技术,对浙江省内枫香自然种群的遗传多样性进行分析。用10个引物对5个枫香种群共100个个体的样品DNA进行扩增,共测得135个位点,其中多态位点为118个,多态位点百分率(P)为87.41%,Shannon指数(I)为0.4646,Nei指数(h)为0.3122,表明枫香总体水平的遗传多样性较高。各种群的多态位点百分率平均为59.11%,Shannon指数平均为0.3660,Nei指数平均为0.2543。P、I、h均显示北山种群最高,天台山种群最低。AMOVA分子差异分析显示:85.49%的变异存在于种群内,14.51%存在于种群间,基因分化系数(Gst)为0.1856,种群间的遗传分化程度较低。种群间的基因流(Nm)为2.1944。5个种群间的平均遗传距离为0.1199。利用UPGMA法对5个种群进行聚类分析,结果分为两大类群:白云山、天台山、北山和安岱后4个种群组成一大类群;大明山种群单独为另一类群。